Non-heme iron (NHFe) enzymes are ubiquitous in biology and catalyze a wide range of reactions involving dioxygen. These include mono- and dioxygenation, H-atom abstraction for hydroxylation, halogenation and desaturation and electrophilic aromatic attack. The non-heme enzymes can be divided into classes that use a ferrous center to activate dioxygen and classes that use a ferric site to activate substrate for the spin forbidden reaction with dioxygen. The ferrous enzymes include the pterin and ?-ketoglutarate dependent dioxygenases, the Rieske dioxygenases, the extradiol dioxygenases and the anticancer drug Bleomycin. While the natures of the cosubstrate-FeII interactions that activate O2 are not defined, peroxo and high valent oxo intermediates have been trapped in a number of these enzymes and relevant model complexes of these intermediates exist with abiological ligation. The ferric/substrate activating enzymes are the lipoxygenases and the intradiol dioxygenases. Lipoxygenase is thought to activate substrate by H-atom abstraction while for the intradiol dioxygenases substrate activation is thought to occur through direct coordination to the ferric center. No intermediate has been trapped for either enzyme. The non-heme iron enzymes have generally been challenging to study due to their lack of spectral features. The goals of this research project have been to generate new spectroscopic methods and approaches to study the non-heme iron enzymes. These methods are used to experimentally define substrate and cofactor interactions with the iron sites and the natures of the intermediates and their key geometric and electronic contributions to reactivity. Further these experimental results are strongly coupled with density functional theory (DFT) calculations to define structure/function correlations, to understand how the iron site is activated for reaction by substrate or cofactor binding, and to understand their reaction mechanisms on a molecular level. Spectroscopic methods are also being developed to directly probe iron centers in highly covalent porphyrin environments to understand how heme relates to NHFe in oxygen activation and in factors that control reactivity.